It has long been suspected that urea accumulation plays a key role in the induction or maintenance of metabolic suppression during extended dormancy in animals from diverse taxa. However, little evidence supporting that hypothesis in living systems exists. We measured aerobic metabolism of isolated organs from the wood frog (Rana sylvatica) in the absence or presence of elevated urea at various temperatures using frogs acclimatized to different seasons. The depressive effect of urea was most prominent in organs from winter and spring frogs. Our results suggest that the presence of urea depresses the metabolism of living organs, and thereby reduces energy expenditure, but its effect varies with temperature and seasonal acclimatization. To investigate the phylogenetic prevalence of urea-induced hypometabolism, we studied four additional species of urea accumulators from the clades Amphibia (Spea bombifrons and Ambystoma tigrinum), Reptilia (Malaclemys terrapin), and Gastropoda (Anguispira alternata), and one amphibian species (Rana pipiens) that does not accumulate urea during dormancy. Urea treatment tended to lower the metabolic rate of isolated organs, as three of the four urea-accumulating species had organs that were responsive to urea treatment. However, organs from R. pipiens, the one species tested that does not accumulate urea during dormancy, were not metabolically affected by urea treatment. Our results further support the hypothesis that urea accumulation can reduce the metabolic rate of dormant animals and provide a rationale for further investigation into the evolution of urea-induced hypometabolism. To better understand the sub-cellular mechanisms underlying urea-induced hypometabolism, we isolated mitochondria from R. sylvatica skeletal muscle, an organ that is metabolically responsive to urea, and measured respiration rates in the absence or presence of elevated urea. Neither state 3 nor state 4 respiration was reduced by urea treatment, suggesting that urea-induced hypometabolism observed at higher levels of organization in hibernating R. sylvatica does not result from direct inhibition of mitochondrial metabolism, and may instead be due to inhibition of energy-utilizing processes elsewhere in the cell.